In addition to tobacco, alcohol is the major risk factor for head and neck cancer. Some of the initial steps of head and neck carcinogenesis are believed to be epigenetic changes, which are defined as changes in gene expression that are not a result of alterations in the underlying DNA coding sequence. Alcohol and its metabolite acetaldehyde (AcH) promote epigenetic changes in breast, liver, and colon cancers, but whether or not genome-wide epigenetic changes are induced by alcohol in oral epithelial cells is unknown. The long term goal of these studies is to understand the molecular mechanisms by which alcohol promotes head and neck tumorigenesis. The objective of this application is to understand whether alcohol regulates genome-wide epigenetic changes, such as DNA methylation and histone modifications in intergenic regions and at the promoters of genes whose expression is changed during human oral carcinogenesis. We will also determine whether retinoic acid (RA) and the DNA methyltransferase inhibitor 5-Aza-2'-deoxycitidine (5-Aza) can reverse epigenetic changes induced by the tobacco carcinogen benzo[a]pyrene (B[a]P) and alcohol. Our central hypothesis is that alcohol and carcinogens found in tobacco will induce genome-wide aberrant epigenetic changes that will alter gene expression of normal human oral epithelial cells, and that retinoic acid and DNA methyltransferase inhibitors will reverse alcohol and carcinogen associated epigenetic changes. Two specific aims will test this hypothesis: Aim 1: To determine if alcohol regulates epigenetic changes and if these changes correlate with changes in gene expression in human oral epithelial cells. Genome-wide sequencing of ethanol treated immortalized OKF6-TERT1 human oral epithelial cells will be performed with the assistance of our institute's Epigenetic Core. This core has expertise in our proposed approaches which include: Reduced Representation Bisulfite Sequencing (RRBS), Chromatin Immunoprecipitation sequencing (ChIP-seq), and RNA-sequencing (RNA-seq) techniques, to determine genome-wide changes in DNA methylation, histone post-translational modifications, and changes in mRNA expression after alcohol treatment, respectively. Aim 2: To determine the role of RA and DNA methyltransferase inhibitors in regulating the epigenome of human oral epithelial cells after alcohol and carcinogen exposure. Cells treated with B[a]P and ethanol will be subjected to RRBS, ChIP-seq, and RNA-seq to determine epigenetic changes and changes in gene expression. We will also examine whether B[a]P and ethanol induced epigenetic changes can be reversed by RA and 5-Aza treatment. This research is innovative because it will use the most cutting edge genome-wide sequencing techniques used to study epigenetic changes. The proposed research is significant as it will fulfill the mission of the National Institute of Dental and Craniofacial Research (NIDCR) and the National Institute of Alcohol Abuse and Alcoholism (NIAAA) by identifying some of the initial steps during carcinogen and alcohol mediated oral carcinogenesis, which will lead to better preventative, prognostic, and treatment approaches for this disease.